Insects have developed unique, innate immmune system. They are able to synthesize a large number of antimicrobial peptides, as a response to invading microorganisms. It is well documented that insects sense foreign entities by cell wall components common in bacteria or fungi, but absent from animals. Recent studies on Galleria mellonella immune system revealed that induction of innate immunity in G. mellonella does not necessary require recognition of microbial cell wall components. The presence of metalloprotease activity (thermolysin-like activity) in hemolymph alone is sufficient to elicit innate immune response in G. mellonella. Many entomopathogenic bacteria, fungi or viruses produce metalloproteases as virulence factors. Secretion of these enzymes into hemolymph during pathogenesis leads to degradation of extracellular matrix protein – collagen IV and releasing peptides (<3kDa) with strong immune stimulatory activity. These peptide fragments activate a signaling pathway leading to expression of immune proteins such as lysozyme and inducible gallerimycin or/and other antimicrobial peptides and IMPI (Insect Metalloproteases Inhibitors).

Our results demonstrated that injection of thermolysin at different, sublethal concentrations (0.1; 0.2; 0.3; 0.4; 0.5 µg/larva) into hemocel of G. mellonella larvae elicited immune response. We observed that lysozyme activity estimated 24h after thermolysin injection increased significantly depending on thermolysin dose. Our studies also revealed that thermolysin injection induced antimicrobial activity against Gram-negative E.coli D31 depending on thermolysin concentration used. Similar results were observed after immunization G. mellonella larvae with heat-killed cells of entomopathogenic strain Pseudomonas aeruginosa, ATCC 27853. Using tricine SDS-PAGE electrophoresis we observed 2 major polypeptide bands below 6.5 kDa after immunization with both thermolysin and microbial elicitors of humoral immune response. These bands present inducible antimicrobial peptides G. mellonella larvae. Injection of thermolysin in sublethal doses induced also thermolysin-inhibitory activity. It is known that immunization G. mellonella larvae with heat-killed cells P. aeruginosa developed protective immunity against subsequent infection with viable cells entomopathogenic strain P. aeruginosa, ATCC 27853. In our experiment we achieved about 46% survival rate for immunized G. mellonella larvae within 48h hours after infection with Pseudomonas aeruginosa. Whereas immune response induced by sublethal doses of thermolysin elicited smaller survival rate, ranging from 15 to 20%, depending on thermolysin dose.

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1. Antimicrobial peptides of Galleria mellonella degraded in vitro and in vivo by Pseudomonas aeruginosa protease B
2. Humoral immune response of Galleria mellonella larvae after infection with entomopathogenic strain Bacillus thuringiensis subsp. kurstaki
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4. Protein kinase A activity in haemocytes of Galleria mellonella larvae upon immune challenge conditions